Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

Abstract

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes.

FIG 1

Rtr1 affects the global distribution of Ser5-P RNAPII. (A) Average gene analysis from ChIP-chip experiments is shown in WT and rtr1Δ strains. Each heat map is divided into quartiles based on gene length, with the 1st quartile representing the shortest 25% of RNAPII target genes and the 4th quartile representing the longest 25% of RNAPII target genes. The color scale of Ser5-P RNAPII intensity is shown to the right. A schematic representation of an average gene is shown at the top. (B) Average gene analysis from median normalized Rpb3-FLAG ChIP-exo experiments is shown from WT and rtr1Δ strains. Each heat map is divided into quartiles based on gene length, with the 1st quartile representing the shortest 25% of RNAPII target genes and the 4th quartile representing the longest 25% of RNAPII target genes. The color scale of total RNAPII occupancy is shown to the right.

FIG 2

ChIP-chip data plotted as the relative enrichment of Ser5-P (Ser5-P in rtr1Δ − Ser5-P in WT cells). Binned Ser5-P RNAPII ChIP-chip data were subjected to k-means clustering, and the average differential enrichment value [rtr1Δ IP − WT IP)/input] for each cluster was plotted. A schematic representation of an average gene is shown at the bottom of the figure. (A) Data from clusters 1 and 3 showing low relative RNAPII Ser5-P occupancy in the coding region of target genes in rtr1Δ strains. (B) Relative Ser5-P RNAPII enrichment in clusters 2 and 4 shows increased Ser5-P levels across the gene (cluster 4) and/or in the 3′ end of the average gene (clusters 2 and 4). (C) Increased levels of Ser5-P RNAPII were observed in rtr1Δ strains at the 5′ ends of cluster 5 genes.

FIG 3

Deletion of RTR1 leads to a genome-wide increase of H3K36me3 at the 3′ ends of coding regions. (A) Average gene analysis from histone H3K36me3 ChIP-chip experiments is shown in WT and rtr1Δ strains following normalization by total histone H3 levels. The heat maps are divided into quartiles based on the transcription rate (Txn rate), with the 1st quartile representing the 25% of yeast genes expressed at low levels and the 4th quartile representing the most highly expressed 25% of yeast genes. The color scale is shown to the right. A schematic representation of an average gene is shown at the top. (B) Average gene analysis from histone H3K36me3 ChIP-chip experiments is shown in WT and rtr1Δ strains following normalization by total histone H3 levels from the 1st quartile and 4th quartile. A schematic of the average gene is shown below the graph.

FIG 4

Changes in Ser5-P RNAPII and histone H3K36me3 levels at PMA1 and LEU1. (A) The relative enrichment of Ser5-P RNAPII occupancy relative to total RNAPII levels (according to Rpb3-FLAG) is shown across a small region of chromosome 7 in WT and rtr1Δ cells. A schematic representation of the PMA1 gene and the downstream LEU1 gene is shown below the graph. The schematic representations of the genes are drawn to scale, and the previously annotated poly(A) sites are indicated by dashed lines. (B) The relative enrichment of H3K6me3 relative to total histone H3 levels is shown across a small region of chromosome 7 in WT and rtr1Δ cells. See the schematic representation of the PMA1 gene and the downstream LEU1 gene for panel A.

FIG 5

A model for the role of Rtr1 in regulating cotranscriptional histone modifications. (Top) Ser5-P is at its highest near the 5′ end of the gene. As polymerase travels through the gene body, Ser2-P begins to increase. These modifications recruit Set2 to methylate coding region histones after the passage of RNAPII. Rtr1 dephosphorylates Ser5-P, essentially “turning off” H3K36me3 prior to the transcription termination site. (Bottom) In an rtr1Δ strain, Set2 retains its affinity for the CTD, and H3K36me3 enrichment is elevated in the coding region and the 3′ end of the gene where the histone mark continues into the transcription termination site at specific RNAPII target genes.